Rat Brainstem Research Articles

External QX-314 inhibits evoked cranial primary afferent synaptic transmission independent of TRPV1.

The cell-impermeant lidocaine derivative QX-314 blocks sodium channels via intracellular mechanisms. In somatosensory nociceptive neurons, open transient receptor potential vanilloid type 1 (TRPV1) receptors provide a transmembrane passageway for QX-314 to produce long-lasting analgesia. Many cranial primary afferents express TRPV1 at synapses on neurons in the nucleus of the solitary tract and caudal trigeminal nucleus (Vc). Here, we investigated whether QX-314 interrupts neurotransmission from primary afferents in rat brain-stem slices. Shocks to the solitary tract (ST) activated highly synchronous evoked excitatory postsynaptic currents (ST-EPSCs). Application of 300 μM QX-314 increased the ST-EPSC latency from TRPV1+ ST afferents, but, surprisingly, it had similar actions at TRPV1- ST afferents. Continued exposure to QX-314 blocked evoked ST-EPSCs at both afferent types. Neither the time to onset of latency changes nor the time to ST-EPSC failure differed between responses for TRPV1+ and TRPV1- inputs. Likewise, the TRPV1 antagonist capsazepine failed to prevent the actions of QX-314. Whereas QX-314 blocked ST-evoked release, the frequency and amplitude of spontaneous EPSCs remained unaltered. In neurons exposed to QX-314, intracellular current injection evoked action potentials suggesting a presynaptic site of action. QX-314 acted similarly at Vc neurons to increase latency and block EPSCs evoked from trigeminal tract afferents. Our results demonstrate that QX-314 blocked nerve conduction in cranial primary afferents without interrupting the glutamate release mechanism or generation of postsynaptic action potentials. The TRPV1 independence suggests that QX-314 either acted extracellularly or more likely entered these axons through an undetermined pathway common to all cranial primary afferents.

Persistent Inflammation-induced Up-regulation of Brain-derived Neurotrophic Factor (BDNF) Promotes Synaptic Delivery of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor GluA1 Subunits in Descending Pain Modulatory Circuits

The enhanced AMPA receptor phosphorylation at GluA1 serine 831 sites in the central pain-modulating system plays a pivotal role in descending pain facilitation after inflammation, but the underlying mechanisms remain unclear. We show here that, in the rat brain stem, in the nucleus raphe magnus, which is a critical relay in the descending pain-modulating system of the brain, persistent inflammatory pain induced by complete Freund adjuvant (CFA) can enhance AMPA receptor-mediated excitatory postsynaptic currents and the GluA2-lacking AMPA receptor-mediated rectification index. Western blot analysis showed an increase in GluA1 phosphorylation at Ser-831 but not at Ser-845. This was accompanied by an increase in distribution of the synaptic GluA1 subunit. In parallel, the level of histone H3 acetylation at bdnf gene promoter regions was reduced significantly 3 days after CFA injection, as indicated by ChIP assays. This was correlated with an increase in BDNF mRNA levels and BDNF protein levels. Sequestering endogenous extracellular BDNF with TrkB-IgG in the nucleus raphe magnus decreased AMPA receptor-mediated synaptic transmission and GluA1 phosphorylation at Ser-831 3 days after CFA injection. Under the same conditions, blockade of TrkB receptor functions, phospholipase C, or PKC impaired GluA1 phosphorylation at Ser-831 and decreased excitatory postsynaptic currents mediated by GluA2-lacking AMPA receptors. Taken together, these results suggest that epigenetic up-regulation of BDNF by peripheral inflammation induces GluR1 phosphorylation at Ser-831 sites through activation of the phospholipase C-PKC signaling cascade, leading to the trafficking of GluA1 to pain-modulating neuronal synapses.

Identification of the pre-Bötzinger complex inspiratory center in calibrated "sandwich" slices from newborn mice with fluorescent Dbx1 interneurons.

Inspiratory active pre‐Bötzinger complex (preBötC) networks produce the neural rhythm that initiates and controls breathing movements. We previously identified the preBötC in the newborn rat brainstem and established anatomically defined transverse slices in which the preBötC remains active when exposed at one surface. This follow‐up study uses a neonatal mouse model in which the preBötC as well as a genetically defined class of respiratory interneurons can be identified and selectively targeted for physiological recordings. The population of glutamatergic interneurons whose precursors express the transcription factor Dbx1 putatively comprises the core respiratory rhythmogenic circuit. Here, we used intersectional mouse genetics to identify the brainstem distribution of Dbx1‐derived neurons in the context of observable respiratory marker structures. This reference brainstem atlas enabled online histology for generating calibrated sandwich slices to identify the preBötC location, which was heretofore unspecified for perinatal mice. Sensitivity to opioids ensured that slice rhythms originated from preBötC neurons and not parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN) cells because opioids depress preBötC, but not pFRG/RTN rhythms. We found that the preBötC is centered ~0.4 mm caudal to the facial motor nucleus in this Cre/lox reporter mouse during postnatal days 0–4. Our findings provide the essential basis for future optically guided electrophysiological and fluorescence imaging‐based studies, as well as the application of other Cre‐dependent tools to record or manipulate respiratory rhythmogenic neurons. These resources will ultimately help elucidate the mechanisms that promote respiratory‐related oscillations of preBötC Dbx1‐derived neurons and thus breathing.

Peripheral injected cholecystokinin-8S modulates the concentration of serotonin in nerve fibers of the rat brainstem

Serotonin and cholecystokinin (CCK) play a role in the short-term inhibition of food intake. It is known that peripheral injection of CCK increases c-Fos-immunoreactivity (Fos-IR) in the nucleus of the solitary tract (NTS) in rats, and injection of the serotonin antagonist ondansetron decreases the number of c-Fos-IR cells in the NTS. This supports the idea of serotonin contributing to the effects of CCK. The aim of the present study was to elucidate whether peripherally injected CCK-8S modulates the concentration of serotonin in brain feeding-regulatory nuclei. Ad libitum fed male Sprague-Dawley rats received 5.2 and 8.7nmol/kg CCK-8S (n=3/group) or 0.15M NaCl (n=3–5/group) injected intraperitoneally (ip). The number of c-Fos-IR neurons, and the fluorescence intensity of serotonin in nerve fibers were assessed in the paraventricular nucleus (PVN), arcuate nucleus (ARC), NTS and dorsal motor nucleus of the vagus (DMV). CCK-8S increased the number of c-Fos-ir neurons in the NTS (mean±SEM: 72±4, and 112±5 neurons/section, respectively) compared to vehicle-treated rats (7±2 neurons/section, P<0.05), but did not modulate c-Fos expression in the DMV or ARC. Additionally, CCK-8S dose-dependently increased the number of c-Fos-positive neurons in the PVN (218±15 and 128±14, respectively vs. 19±5, P<0.05). In the NTS and DMV we observed a decrease of serotonin-immunoreactivity 90min after injection of CCK-8S (46±2 and 49±8 pixel/section, respectively) compared to vehicle (81±8 pixel/section, P<0.05). No changes of serotonin-immunoreactivity were observed in the PVN and ARC. Our results suggest that serotonin is involved in the mediation of CCK-8's effects in the brainstem.

Restoration of glutamine synthetase activity, nitric oxide levels and amelioration of oxidative stress by propolis in kainic acid mediated excitotoxicity.

Propolis has been proposed to be protective on neurodegenerative disorders. To understand the neuroprotective effects of honeybee propolis, glutamine synthetase (GS) activity, nitric oxide (NO), thiobarbituric acid reactive substances (TBARS) and total antioxidant status (TAS) were studied in different brain regions-cerebral cortex (CC), cerebellum (CB) and brain stem (BS) of rats supplemented with propolis and subjected to kainic acid (KA) mediated excitotoxicity. Male Sprague-Dawley rats were divided into four groups; Control group and KA group received vehicle and saline. Propolis group and propolis + KA group were orally administered with propolis (150mg/kg body weight), five times every 12 hours. KA group and propolis + KA group were injected subcutaneously with kainic acid (15mg/kg body weight) and were sacrificed after 2 hrs and CC, CB and BS were separated homogenized and used for estimation of GS activity, NO, TBARS, and TAS concentrations by colorimetric methods. Results were analyzed by one-way ANOVA, reported as mean + SD from 6 animals, and p<0.05 considered statistically significant. NO was increased (p< 0.001) and GS activity was decreased (p< 0.001) in KA treated group compared to control group as well as propolis + KA treated group. TBARS was decreased and TAS was increased (p< 0.001) in propolis + KA treated group compared KA treated group. This study clearly demonstrated the restoration of GS activity, NO levels and decreased oxidative stress by propolis in kainic acid mediated excitotoxicity. Hence the propolis can be a possible potential candidate (protective agent) against excitotoxicity and neurodegenerative disorders.

Correction: Unacylated Ghrelin Suppresses Ghrelin-Induced Neuronal Activity in the Hypothalamus and Brainstem of Male Rats

Correction: Unacylated Ghrelin Suppresses Ghrelin-Induced Neuronal Activity in the Hypothalamus and Brainstem of Male Rats

A method to make a craniotomy on the ventral skull of neonate rodents.

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

Unacylated ghrelin suppresses ghrelin-induced neuronal activity in the hypothalamus and brainstem of male rats [corrected

Ghrelin, the endogenous growth hormone secretagogue, has an important role in metabolic homeostasis. It exists in two major molecular forms: acylated (AG) and unacylated (UAG). Many studies suggest different roles for these two forms of ghrelin in energy balance regulation. In the present study, we compared the effects of acute intracerebroventricular administration of AG, UAG and their combination (AG+UAG) to young adult Wistar rats on food intake and central melanocortin system modulation. Although UAG did not affect food intake it significantly increased the number of c-Fos positive neurons in the arcuate (ARC), paraventricular (PVN) and solitary tract (NTS) nuclei. In contrast, UAG suppressed AG-induced neuronal activity in PVN and NTS. Central UAG also modulated hypothalamic expression of Mc4r and Bmp8b, which were increased and Mc3r, Pomc, Agrp and Ucp2, which were decreased. Finally, UAG, AG and combination treatments caused activation of c-Fos in POMC expressing neurons in the arcuate, substantiating a physiologic effect of these peptides on the central melanocortin system. Together, these results demonstrate that UAG can act directly to increase neuronal activity in the hypothalamus and is able to counteract AG-induced neuronal activity in the PVN and NTS. UAG also modulates expression of members of the melanocortin signaling system in the hypothalamus. In the absence of an effect on energy intake, these findings indicate that UAG could affect energy homeostasis by modulation of the central melanocortin system.

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

The expression of SIRT1 in brainstem of rats in different types of hypoxia and its significance

To explore the correlation between SIRT1 expression in brainstem and apnea hypopnea index and the effect of hypoxia on SIRT1 expression in rats. Thirty-three healthy Sprague-Dawley(SD) rats underwent sleep monitoring and SIRT1 protein in brainstems was measured by Western blot. The correlation of sleep apnea index and SIRT1 protein level in brainstem was analysed. Another 36 adult male SD rats were randomly and equally divided into 4 groups: normal control (NC) group, continuous hypoxia (CH) group, intermittent hypoxia (IH) group and intermittent hypoxia with hypercapnia (IHH) group. The expression of SIRT1 protein of the lower brainstems of the 4 groups were measured by Western blot. The level of SIRT1 in the brainstem was negatively correlated with the frequency of spontaneous sleep apnea in REM(r = -0.617, P < 0.001) while positively correlated with the frequency of post sigh sleep apnea in REM (r = 0.535, P < 0.01). The relative expression level of SIRT1 in NC, CH, IH, IHH group were (1.4 ± 1.0), (1.4 ± 0.6), (2.5 ± 1.0) , (1.6 ± 1.2), respectively. There was a significant difference in the SIRT1 level among groups (F = 2.964, P < 0.05) . The rats with lower level of SIRT1 may have lower anti-oxidative stress capability which results in higher frequency of spontaneous sleep apnea. Under the circumstance of hypoxia, the fact that the expression of SIRT1 protein was elevated may be a protective mechanism.

Tu1793 Elevated Sympathetic Activity Induces Anxiety-Like Behavior in Rats Subjected to Neonatal Inflammation

G A A b st ra ct s neurons. In contrast to control rats, UK14304 decreased the amplitude of IPSC in 12 of 17 rats that underwent homotypic stress (2hrs restrain, 20min forced swim, 40min cold @4°C and 2hrs water avoidance) for 5 days in a concentration-dependent manner (91±1%, 78±1% and 53±9% of baseline amplitude in response to perfusion with 0.1, 1 and 3μM, respectively; P<0.05 vs control). Pretreatment with CRF had no additional inhibitory action on the UK14304-induced inhibition of GABAergic currents. Miniature strain gauges were applied to the anterior corpus of anaesthetized rats to record gastric motility. In control rats, DMV microinjections of the indirect sympathomimetic tyramine (4.5nmoles/60nl) reduced gastric motility to 50±10% of baseline (N=10); conversely, microinjections of tyramine in the brainstem of homotypic stress rats decreased gastric motility to 7±5% of baseline (N=2). These data suggest that homotypic stress induces an upregulation of catecholaminergic, including α2-mediated, responses in vagal neurocircuits resulting in an exaggerated vagallymediated gastroinhibition. Supported by DK55530 and DK78364

Cystatin C Has a Dual Role in Post-Traumatic Brain Injury Recovery

Cathepsin B is one of the major lysosomal cysteine proteases involved in neuronal protein catabolism. This cathepsin is released after traumatic injury and increases neuronal death; however, release of cystatin C, a cathepsin inhibitor, appears to be a self-protective brain response. Here we describe the effect of cystatin C intracerebroventricular administration in rats prior to inducing a traumatic brain injury. We observed that cystatin C injection caused a dual response in post-traumatic brain injury recovery: higher doses (350 fmoles) increased bleeding and mortality, whereas lower doses (3.5 to 35 fmoles) decreased bleeding, neuronal damage and mortality. We also analyzed the expression of cathepsin B and cystatin C in the brains of control rats and of rats after a traumatic brain injury. Cathepsin B was detected in the brain stem, cerebellum, hippocampus and cerebral cortex of control rats. Cystatin C was localized to the choroid plexus, brain stem and cerebellum of control rats. Twenty-four hours after traumatic brain injury, we observed changes in both the expression and localization of both proteins in the cerebral cortex, hippocampus and brain stem. An early increase and intralysosomal expression of cystatin C after brain injury was associated with reduced neuronal damage.

Vagally‐mediated gastric effects of catecholamines in stressed rats (1110.13)

Stress exerts vagally‐mediated action to disrupt gastric motility. Preliminary results from our laboratory suggest that brainstem vagal neurocircuits undergo remodeling following stress and previous studies from other groups have shown that the A2 catecholaminergic area is involved in these effects.The aim of this study was to investigate whether stress modulates the response of vagally‐mediated gastric pathways to catecholamines.Inhibitory postsynaptic currents (IPSCs) were recorded in neurons of the dorsal motor nucleus of the vagus (DMV) following NTS stimulation and perfusion of the slices with the α2 agonist Uk14304 (0.1‐10μM). In control rats, UK14304 had no effect on 5 of 5 neurons tested (96±3% of baseline amplitude); in contrast, UK14304 decreased the amplitude of IPSC in 12 of 17 rats that underwent homotypic stress for 5 days in a concentration‐dependent manner (91±1%, 78±1% and 53±9% of baseline amplitude in response to 0.1, 1 and 3μM, respectively; P&lt;0.05 vs control). In control rats, brainstem microinjections of the indirect sympathomimetic tyramine (4.5nmoles/60nl) reduced gastric motility to 50±10% of baseline (N=10); conversely, microinjections of tyramine in the brainstem of homotypic stress rats decreased gastric motility to 7±5% of baseline (N=2).These data suggest that homotypic stress induces an upregulation of catecholaminergic, including α2‐mediated, responses in vagal neurocircuits resulting in an exaggerated vagally‐mediated gastroinhibition.Grant Funding Source: NIH DK55530, DK78364

Hydrogen peroxide modulates barium‐sensitive K+‐currents of nucleus tractus solitarii neurons (686.14)

The nTS holds a vital role in cardiorespiratory reflexes as the first central integration site of chemosensitive afferents. Hypoxia‐reoxygenation over‐produces H2O2, a potent neuromodulator and reactive oxygen species. We previously found in rat brainstem slices that second‐order nTS neurons initially reduce excitability when exposed to 500 µM H2O2. This was due to a reversible hyperpolarization in resting membrane potential (RMP; ‐10 mV) and reduction in membrane resistance (Ri; ‐224 MΩ). Since these effects occur at RMP (~ ‐60 mV), we determined the effect of H2O2 on K+ channel conductance using voltage steps and ramps between ‐100 and ‐50 mV in nTS slices. H2O2 increased the slope of holding currents during steps by 1.31 ±0.35 nS (N=6, p蠄0.01). Such currents reversed at ‐75 mV, indicating greater K+ conductance. Augmented conductance (+ 0.49 ±0.18 nS; N=5, p蠄0.05) by H2O2 was also observed during voltage ramps. Conductance changes persisted during blockade of glutamate and GABAA receptors, confirming such effects were not secondary to synaptic activation. H2O2‐induced alterations of RMP, Ri and slope conductance were eliminated by barium (100 µM), a blocker of inward rectifying (IRK) and 2‐pore‐domain (K2P) K+ channels. Altogether, the H2O2 induced increase in barium‐sensitive K+ conductance is likely an immediate response to increased ROS and mediates decreased neuronal activity.Grant Funding Source: Supported by: RO1 HL098602, AHA 12POST11670002 (TDO).

Development of respiratory pattern in mice (1177.1)

Maturation of mouse respiratory pattern in vivo has been studied (Euro J Neurosci 17:1233, 2003). The results, however, were grouped at P0‐P2. In vitro recordings from en block late embryonic rat brainstem spinal cord preparation showed prolonged neuronal activity in cervical ventral root 5 bursts (J Neurophysiol 67:996, 1992). Here we show in C57BL6/J mice a change in respiratory pattern from P0.5 to P2. Studies were performed with body plethysmography at ~32°C. This system (J Appl Physiol 56:1533, 1984) determines inspiratory and expiratory flow. At P0.5 (wt=1.38±0.08g) TI showed a sustained plateau pattern lasting 253±17 ms. TE (173±14 ms) was shorter resulting in a duty cycle (TI/TTOT) of 0.59±0.01. Respiratory frequency increased from 145±9 bpm at P0.5 to 188±19 at P2 (wt=1.73±.05g). The increase in frequency was entirely due to a change in TI that decreased from 253±17 to 135±11 ms. Inspiratory flow no longer showed a plateau during most breaths. TE remained constant over the first 2 postnatal days (173±14 vs 208±22 ms). TI/TTOT was reduced from 0.59±0.01 to 0.40±0.01. The results suggest that pontine pneumotactic centre and/or its afferent inputs are no fully mature in P0.5 mice.Grant Funding Source: Supported by Rett Syndrome Research Trust

Tapentadol inhibits calcitonin gene-related peptide release from rat brainstem in vitro

We have previously developed an in vitro model of rat brainstem explants. The latter release sizable amounts of calcitonin gene-related peptide (CGRP); basal release can be stimulated by such secretagogues as high KCl concentrations, veratridine or capsaicine. In this paradigm we investigated the activity of the analgesic agent tapentadol; the effects of tapentadol were compared to those of a classical opioid receptor agonist, morphine, and the selective noradrenaline reuptake inhibitor reboxetine. Morphine inhibited basal CGRP release, with statistical significance from 1nM onward and maximal (−44%) inhibition at 100μM. Morphine also inhibited K+-stimulated peptide release, with a significant effect from 1μM and maximal (−39%) decrease at 100μM, but failed to inhibit release stimulated by 10μM capsaicin. At variance, reboxetine had no effect on baseline CGRP outflow, but was able to inhibit both K+-stimulated [significant inhibition from 1μM onward and maximal (−37%) decrease at 100μM], and capsaicin-stimulated release [significant effect from 1μM and maximal (−31%) decrease at 100μM]. Likewise, tapentadol had no effect on baseline CGRP release up to 100μM, but decreased secretion stimulated by 56mM KCl or capsaicin, with significant effects from 0.1 and 1μM respectively; maximal inhibition over 56mM KCl and capsaicin stimuli was −29% and −31%, respectively. Naloxone antagonized the effect of morphine, but not those of reboxetine and tapentadol, on K+-stimulated CGRP secretion. In conclusion the present study provides consistent pharmacological evidence that tapentadol acts as a noradrenaline reuptake inhibitor agent in this experimental model.

The effect of air puff stress on c‐Fos expression in rat hypothalamus and brainstem: central circuitry mediating sympathoexcitation and baroreflex resetting

Psychological stress evokes increases in sympathetic activity and blood pressure, which are due at least in part to an upward resetting of the baroreceptor-sympathetic reflex. In this study we determined whether sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM), which have a critical role in the reflex control of sympathetic activity, are activated during air puff stress, a moderate psychological stressor. Secondly, we identified neurons that are activated by air puff stress and that also project to the nucleus tractus solitarius (NTS), a key site for modulation of the baroreceptor reflex. Air puff stress resulted in increased c-Fos expression in several hypothalamic and brainstem nuclei, including the paraventricular nucleus (PVN), dorsomedial hypothalamus, perifornical area (PeF), periaqueductal gray (PAG), NTS and rostral ventromedial medulla, but not in the RVLM region that contains sympathetic premotor neurons. In contrast, neurons in this RVLM region, including catecholamine-synthesizing neurons, did express c-Fos following induced hypotension, which reflexly activates RVLM sympathetic premotor neurons. The highest proportion of NTS-projecting neurons that were double-labelled with c-Fos after air puff stress was in the ventrolateral PAG (29.3 ± 5.5%), with smaller but still significant proportions of double-labelled NTS-projecting neurons in the PVN and PeF (6.5 ± 1.8 and 6.4 ± 1.7%, respectively). The results suggest that the increased sympathetic activity during psychological stress is not driven primarily by RVLM sympathetic premotor neurons, and that neurons in the PVN, PeF and ventrolateral PAG may contribute to the resetting of the baroreceptor-sympathetic reflex that is associated with psychological stress.

5-Hydroxytryptamine–Mediated Neurotransmission Modulates Spontaneous and Vagal-Evoked Glutamate Release in the Nucleus of the Solitary Tract Effect of Uptake Blockade

The effect of blockade of either 5-hydroxytryptamine (5-HT)/serotonin transporter (SERT) with citalopram or the organic cation transporter 3 (OCT3)/plasma membrane monoamine transporter (PMAT) with decynium-22 (D-22) on spontaneous and evoked release of 5-HT in the nucleus tractus solitarius (NTS) was investigated in rat brainstem slices treated with gabazine. 5-HT release was measured indirectly by changes in the frequency and amplitude of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) [in the presence of tetrodotoxin (TTX)] and evoked EPSCs. Blockade of 5-HT3 receptors with granisetron reduced, whereas the 5-HT3 agonist phenylbiguanide increased, the frequency of mEPSCs. 5-HT decreased mEPSC frequency at low concentrations and increased frequency at high concentrations. This inhibition was blocked by the 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide (WAY-100635), which was ineffective on its own, whereas the excitation was reversed by granisetron. The addition of citalopram or D-22 caused inhibition, which was prevented by 5-HT1A blockade. Thus, in the NTS, the spontaneous release of 5-HT is able to activate 5-HT3 receptors, but not 5-HT1A receptors, as the release in their vicinity is removed by uptake. The ineffectiveness of corticosterone suggests that the low-affinity, high-capacity transporter is PMAT, not OCT3. For evoked 5-HT release, only D-22 caused an increase in the amplitude of EPSCs, with a decrease in the paired pulse ratio, and increased the number of spontaneous EPSCs after 20-Hz stimulation. Thus, for the evoked release of 5-HT, the low-affinity, high-capacity transporter PMAT, but not 5-HT transporter (5-HTT)/SERT, is important in the regulation of changes in 5-HT extracellular concentration.

Hypothyroidism alters antioxidant defence system in rat brainstem during postnatal development and adulthood

The present investigation was carried out to evaluate alterations in oxidative stress parameter [lipid peroxidation (LPx)] and antioxidant enzyme activities [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] in rat brainstem in response to neonatal hypothyroidism during development (from birth to 7, 15 and 30 days old) and adulthood (90 days old). Hypothyroidism in rats was induced by feeding the lactating mothers (from the day of parturition till weaning, 25 days old) or directly to the pups with 0.05 % [6-n-propyl 2-thiouracil (PTU)] in drinking water. Increased level of LPx was observed in brainstem of 7 days old hypothyroid rats, accompanied by augmented activities of SOD and GPx. In 15 and 30 days old hypothyroid rat brainstem, a significant decline in LPx was observed. Significantly increased activities of CAT and GPx were observed in 15 and 30 days PTU-treated rats. Decreased level of LPx was observed in brainstem of rats treated with PTU from birth to 30 days followed by withdrawal up to 90 days of age (transient hypothyroidism) as compared to control and persistent treatment of PTU up to 90 days of age. Activities of CAT and GPx were decreased in persistent hypothyroid rats of 90 days old with respect to control and transient hypothyroid rats. On the other hand, SOD activity was decreased in both persistent and transient hypothyroid rats with respect to control rats. These results suggest that the PTU-induced neonatal hypothyroidism modulates the antioxidant defence system during postnatal development and adulthood in brainstem of rats.

The Yin and Yang of Calcium Effects on Synaptic Vesicle Endocytosis

A large number of studies suggest that calcium triggers and accelerates vesicle endocytosis at many synapses and non-neuronal secretory cells. However, many studies show that prolonging the duration of the stimulation train, which induces more calcium influx, slows down endocytosis; and several studies suggest that instead of triggering endocytosis, calcium actually inhibits endocytosis. Here we addressed this apparent conflict at a large nerve terminal, the calyx of Held in rat brainstem, in which recent studies suggest that transient calcium increase up to tens of micromolar concentration at the micro/nano domain triggers endocytosis. By dialyzing 0-1 μM calcium into the calyx via a whole-cell pipette, we found that slow endocytosis was inhibited by calcium dialysis in a concentration-dependent manner. Thus, prolonged, small, and global calcium increase inhibits endocytosis, whereas transient and large calcium increase at the micro/nano domain triggers endocytosis and facilitates endocytosis. This yin and yang effect of calcium may reconcile apparent conflicts regarding whether calcium accelerates or inhibits endocytosis. Whether endocytosis is fast or slow depends on the net outcome between the yin and yang effect of calcium.